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ABSTRACT: Photoreceptor terminals contain post-synaptic density (PSD) proteins e.g., PSD-95/PSD-93, but their role at photoreceptor synapses is not known. PSDs are generally restricted to post-synaptic boutons in central neurons and form scaffolding with multiple proteins that have structural and functional roles in neuronal signaling. The Shank family of proteins (Shank 1-3) functions as putative anchoring proteins for PSDs and is involved in the organization of cytoskeletal/signaling complexes in neurons. Specifically, Shank 1 is restricted to neurons and interacts with both receptors and signaling molecules at central neurons to regulate plasticity. However, it is not known whether Shank 1 is expressed at photoreceptor terminals. In this study we have investigated Shank 1A localization in the outer retina at photoreceptor terminals. We find that Shank 1A is expressed presynaptically in cone pedicles, but not rod spherules, and it is absent from mice in which the Shank 1 gene is deleted. Shank 1A co-localizes with PSD-95, peanut agglutinin, a marker of cone terminals, and glycogen phosphorylase, a cone specific marker. These findings provide convincing evidence for Shank 1A expression in both the inner and outer plexiform layers, and indicate a potential role for PSD-95/Shank 1 complexes at cone synapses in the outer retina.
PLoS ONE 01/2012; 7(9):e43463. · 4.09 Impact Factor
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ABSTRACT: Vesicle release from photoreceptor ribbon synapses is regulated by L-type Ca(2+) channels, which are in turn regulated by Cl(-) moving through calcium-activated chloride [Cl(Ca)] channels. We assessed the proximity of Ca(2+) channels to release sites and Cl(Ca) channels in synaptic terminals of salamander photoreceptors by comparing fast (BAPTA) and slow (EGTA) intracellular Ca(2+) buffers. BAPTA did not fully block synaptic release, indicating some release sites are <100 nm from Ca(2+) channels. Comparing Cl(Ca) currents with predicted Ca(2+) diffusion profiles suggested that Cl(Ca) and Ca(2+) channels average a few hundred nanometers apart, but the inability of BAPTA to block Cl(Ca) currents completely suggested some channels are much closer together. Diffuse immunolabeling of terminals with an antibody to the putative Cl(Ca) channel TMEM16A supports the idea that Cl(Ca) channels are dispersed throughout the presynaptic terminal, in contrast with clustering of Ca(2+) channels near ribbons. Cl(Ca) currents evoked by intracellular calcium ion concentration ([Ca(2+)](i)) elevation through flash photolysis of DM-nitrophen exhibited EC(50) values of 556 and 377 nM with Hill slopes of 1.8 and 2.4 in rods and cones, respectively. These relationships were used to estimate average submembrane [Ca(2+)](i) in photoreceptor terminals. Consistent with control of exocytosis by [Ca(2+)] nanodomains near Ca(2+) channels, average submembrane [Ca(2+)](i) remained below the vesicle release threshold (∼ 400 nM) over much of the physiological voltage range for cones. Positioning Ca(2+) channels near release sites may improve fidelity in converting voltage changes to synaptic release. A diffuse distribution of Cl(Ca) channels may allow Ca(2+) influx at one site to influence relatively distant Ca(2+) channels.
Journal of Neurophysiology 11/2010; 105(1):321-35. · 3.32 Impact Factor
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ABSTRACT: Gamma-aminobutyric acid (GABA) is likely expressed in horizontal cells of all species, although conflicting physiological findings have led to considerable controversy regarding its role as a transmitter in the outer retina. This study has evaluated key components of the GABA system in the outer retina of guinea pig, an emerging retinal model system. The presence of GABA, its rate-limiting synthetic enzyme glutamic acid decarboxylase (GAD(65) and GAD(67) isoforms), the plasma membrane GABA transporters (GAT-1 and GAT-3), and the vesicular GABA transporter (VGAT) was evaluated by using immunohistochemistry with well-characterized antibodies. The presence of GAD(65) mRNA was also evaluated by using laser capture microdissection and reverse transcriptase-polymerase chain reaction. Specific GABA, GAD(65), and VGAT immunostaining was localized to horizontal cell bodies, as well as to their processes and tips in the outer plexiform layer. Furthermore, immunostaining of retinal whole mounts and acutely dissociated retinas showed GAD(65) and VGAT immunoreactivity in both A-type and B-type horizontal cells. However, these cells did not contain GAD(67), GAT-1, or GAT-3 immunoreactivity. GAD(65) mRNA was detected in horizontal cells, and sequencing of the amplified GAD(65) fragment showed approximately 85% identity with other mammalian GAD(65) mRNAs. These studies demonstrate the presence of GABA, GAD(65), and VGAT in horizontal cells of the guinea pig retina, and support the idea that GABA is synthesized from GAD(65), taken up into synaptic vesicles by VGAT, and likely released by a vesicular mechanism from horizontal cells.
The Journal of Comparative Neurology 12/2009; 518(10):1647-69. · 3.81 Impact Factor
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ABSTRACT: In the retina, adenosine is released in the dark and has been shown to inhibit Ca2+ influx through voltage-gated Ca2+ channels in cones. Therefore, we tested whether adenosine can inhibit exocytosis from isolated cone photoreceptors. Simultaneous measurements of membrane exocytosis and Ca2+ were made from cones using the activity-dependent dye, Synaptored-C2, and the Ca2+ indicator dye, Fluo-4. Adenosine suppressed exocytosis in cones, indicating that transmitter release is also reduced from cone terminals, and further supports an inhibitory mechanism for modulating transmitter release onto second-order neurons. Furthermore, this raises the possibility that adenosine might be neuroprotective for photoreceptors and second-order neurons by suppressing Ca2+ levels in cones and reducing exocytosis of L-glutamate, respectively.
Neuroreport 08/2009; 20(10):923-9. · 1.66 Impact Factor
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ABSTRACT: The global disparity in cancer incidence remains a major public health problem. We focused on prostate cancer since microscopic disease in men is common, but the incidence of clinical disease varies more than 100 fold worldwide. Ca(2+) signaling is a central regulator of cell proliferation, but has received little attention in cancer prevention. We and others have reported a strong dose-dependent reduction in the incidence of prostate and lung cancer within populations exposed to boron (B) in drinking water and food; and in tumor and cell proliferation in animal and cell culture models.
We examined the impact of B on Ca(2+) stores using cancer and non-cancer human prostate cell lines, Ca(2+) indicators Rhod-2 AM and Indo-1 AM and confocal microscopy. In DU-145 cells, inhibition of Ca(2+) release was apparent following treatment with Ringers containing RyR agonists cADPR, 4CmC or caffeine and respective levels of BA (50 microM), (1, 10 microM) or (10, 20, 50,150 microM). Less aggressive LNCaP cancer cells required 20 microM BA and the non-tumor cell line PWR1E required 150 microM BA to significantly inhibit caffeine stimulated Ca(2+) release. BA (10 microM) and the RyR antagonist dantroline (10 microM) were equivalent in their ability to inhibit ER Ca(2+) loss. Flow cytometry and confocal microscopy analysis showed exposure of DU-145 cells to 50 microM BA for 1 hr decreased stored [Ca(2+)] by 32%.
We show B causes a dose dependent decrease of Ca(2+) release from ryanodine receptor sensitive stores. This occurred at BA concentrations present in blood of geographically disparate populations. Our results suggest higher BA blood levels lower the risk of prostate cancer by reducing intracellular Ca(2+) signals and storage.
PLoS ONE 02/2009; 4(6):e6009. · 4.09 Impact Factor
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ABSTRACT: Immunoreactivity for the plasma membrane GABA transporter, GAT-3 (red), in a vertical section of adult mouse retina, with the Müller glia identified with CRALBP antibodies (green) and bipolar cell and some Müller cell somata with Chx10 antibodies (blue). J. Comp. Neurol. 512:6-26, 2009. (c) 2008 Wiley-Liss, Inc.
The Journal of Comparative Neurology 12/2008; 512(2):spc1. · 3.81 Impact Factor
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ABSTRACT: Galanin activates three receptors, the galanin receptor 1 (GalR1), GalR2, and GalR3. In the gastrointestinal tract, GalR1 mediates the galanin inhibition of cholinergic transmission to the longitudinal muscle and reduction of peristalsis efficiency in the small intestine. Galanin has also been shown to inhibit depolarization-evoked Ca2+ increases in cultured myenteric neurons. Because GalR1 immunoreactivity is localized to cholinergic myenteric neurons, we hypothesized that this inhibitory action of galanin on myenteric neurons is mediated by GalR1. We investigated the effect of galanin 1-16, which has high affinity for GalR1 and GalR2, in the presence or absence of the selective GalR1 antagonist, RWJ-57408, and of galanin 2-11, which has high affinity for GalR2 and GalR3, on Ca2+ influx through voltage-dependent Ca2+ channels in cultured myenteric neurons. Myenteric neurons were loaded with fluo-4 and depolarized by high K+ concentration to activate voltage-dependent Ca2+ channels. Intracellular Ca2+ levels were quantified with confocal microscopy. Galanin 1-16 (0.01-1 microM) inhibited the depolarization-evoked Ca2+ increase in a dose-dependent manner with an EC(50) of 0.172 microM. The selective GalR1 antagonist, RWJ-57408 (10 microM), blocked the galanin 1-16 (1 microM)-mediated inhibition of voltage-dependent Ca2+ channel. By contrast, the GalR2/GalR3 agonist, galanin 2-11 did not affect the K+-evoked Ca2+ influx in myenteric neurons. GalR1 immunoreactivity was localized solely to myenteric neurons in culture, as previously observed in intact tissue. These findings indicate that the inhibition of depolarization-evoked Ca2+ influx in myenteric neurons in culture is mediated by GalR1 and confirm the presence of functional GalR1 in the myenteric plexus. This is consonant with the hypothesis that GalR1 mediates galanin inhibition of transmitter release from myenteric neurons.
Journal of Neuroscience Research 12/2008; 87(5):1107-14. · 2.74 Impact Factor
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ABSTRACT: Glutamate is the major excitatory neurotransmitter in the retina, and most glutamatergic neurons express one of the three known vesicular glutamate transporters (VGLUT1, 2, or 3). However, the expression profiles of these transporters vary greatly in the retina. VGLUT1 is expressed by photoreceptor and bipolar cell terminals, and VGLUT2 appears to be predominately expressed by ganglion cells, and perhaps Müller cells, cone photoreceptor terminals, and horizontal cells in some species. The discovery of a third vesicular glutamate transporter, VGLUT3, has brought about speculation concerning its role and function based on its expression in amacrine cells. To address this we studied the postnatal development of VGLUT3 from day 0 through adult in the rat retina, and compared this with the expression patterns of VGLUT1 and VGLUT2. VGLUT3 expression was restricted to a population of amacrine cells. Expression of VGLUT3 was first observed at postnatal day 10 (P10) in the soma and some processes, which extensively arborized in both the ON and OFF sublamina of the IPL by P15. In contrast, VGLUT1 and VGLUT2 expression appeared earlier than VGLUT3; with VGLUT1 initially detected at P5 in photoreceptor terminals and P6 in bipolar terminals, and VGLUT2 immunoreactivity initially detected at P0 in ganglion cell bodies, and remained prominent throughout all stages of development. Interestingly, VGLUT3 has extensive somatic expression throughout development, which could be involved in non-synaptic modulation by glutamate in developing retina, and could influence trophic and extra-synaptic neuronal signaling by glutamate in the inner retina.
Brain Research 07/2008; 1215:20-9. · 2.73 Impact Factor
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ABSTRACT: Endogenous adenosine has already been shown to inhibit transmitter release from the rod synapse by suppressing Ca(2+) influx through voltage-gated Ca(2+) channels. However, it is not clear how adenosine modulates the cone synapse. Cone photoreceptors, like rod photoreceptors, also possess L-type Ca(2+) channels that regulate the release of L-glutamate. To assess the impact of adenosine on Ca(2+) influx though voltage-gated Ca(2+) channels in cone terminals, whole-cell perforated-patch clamp recording and Ca(2+) imaging with fluo-4 were used on isolated cones and salamander retinal slices. Synaptic markers (VAMP and piccolo) and activity-dependent dye labeling revealed that tiger salamander cone terminals contain a broad, vesicle-filled cytoplasmic extension at the base of the somatic compartment, which is unlike rod terminals that contain one or more thin axons, each terminating in a large bulbous synaptic terminal. The spatiotemporal Ca(2+) responses of the cone terminals do not differ significantly from the Ca(2+) responses of the soma or inner segment like that observed in rods. Whole-cell recording of cone I(Ca) and Ca(2+) imaging of synaptic terminals in cones demonstrate that adenosine inhibited both I(Ca) and the depolarization-evoked Ca(2+) increase in cone terminals in a dose-dependent manner from 1 to 50 muM. These results indicate that, as in rods, adenosine's ability to suppress voltage-dependent Ca(2+) channels at the cone synapse will limit the amount of L-glutamate released. Therefore, adenosine has an inhibitory effect on L-glutamate release at the first synapse, which likely favors elevated adenosine levels in the dark or during dark-adapted conditions.
Journal of Neuroscience Research 05/2007; 85(5):1126-37. · 2.74 Impact Factor
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ABSTRACT: Galanin functions are mediated by three distinct G-protein-coupled receptors, galanin receptor 1 (GalR1), GalR2 and GalR3, which activate different intracellular signaling pathways. Here, we quantified mRNA levels of GalR1, GalR2 and GalR3 in the gastrointestinal tract using real time RT-PCR. GalR1 and GalR2 mRNAs were detected in all segments with the highest levels in the large intestine and stomach, respectively. GalR3 mRNA levels were quite low and mostly confined to the colon. We also investigated the effect of galanin 1-16, which has high affinity for GalR1 and GalR2 and low affinity for GalR3 on depolarization-evoked Ca2+ increases in rat cultured myenteric neurons using Ca2+-imaging. Intracellular Ca2+ changes in myenteric neurons were monitored using the Ca2+ sensitive dye, fluo-4, and confocal microscopy. Galanin 1-16 (1 microM) markedly inhibited the K+-evoked Ca2+ increases in myenteric neurons. In summary, the differential distribution of GalRs supports the hypothesis that the complex effects of galanin in the gastrointestinal tract result from the activation of multiple receptor subtypes. Furthermore, this study confirms the presence of functional GalRs and suggests that galanin modulates transmitter release from myenteric neurons through inhibition of voltage-dependent calcium channels involving a G(i/o)-coupled GalR.
Neuropeptides 07/2005; 39(3):349-52. · 1.55 Impact Factor
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ABSTRACT: Adenosine is released from retina in darkness; photoreceptors possess A2 adenosine receptors, and A2 agonists inhibit L-type Ca2+ currents (ICa) in rods. We therefore investigated whether A2 agonists inhibit rod inputs into second-order neurons and whether selective antagonists to A1, A2A, or A3 receptors prevent Ca2+ influx through rod ICa. [Ca2+]i changes in rods were assessed with fura-2. ICa in rods and light responses of rods and second-order neurons were recorded using perforated patch-clamp techniques in the aquatic tiger salamander retinal slice preparation. Consistent with earlier results using the A2 agonist N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA), the A2A agonist CGS-21680 significantly inhibited ICa and depolarization-evoked [Ca2+]i increases in rods. The A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and A2A antagonist, ZM-241385, but not the A3 antagonist, VUF-5574, inhibited effects of adenosine on Ca2+ influx in rods. DPCPX and ZM-241385 also inhibited effects of CGS-21680, suggesting they both act at A2A receptors. Both A2 agonists, CGS-21680 and DPMA, reduced light-evoked currents in second-order neurons but not light-evoked voltage responses of rods, suggesting that activation of A2 receptors inhibits transmitter release from rods. The inhibitory effects of CGS-21680 on both depolarization-evoked Ca2+ influx and light-evoked currents in second-order neurons were antagonized by ZM-241385. By itself, ZM-241385 enhanced the light-evoked currents in second-order neurons, suggesting that endogenous levels of adenosine inhibit transmitter release from rods. The effects of these drugs suggest that endogenous adenosine activates an A2-like adenosine receptor on rods leading to inhibition of ICa, which in turn inhibits l-glutamate release from rod photoreceptors.
Journal of Neurophysiology 08/2003; 90(1):165-74. · 3.32 Impact Factor
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ABSTRACT: Presynaptic inhibition is a major mechanism for regulating synaptic transmission in the CNS and adenosine inhibits Ca(2+) currents (I(Ca)) to reduce transmitter release at several synapses. Rod photoreceptors possess L-type Ca(2+) channels that regulate the release of L-glutamate. In the retina, adenosine is released in the dark when L-glutamate release is maximal. We tested whether adenosine inhibits I(Ca) and intracellular Ca(2+) increases in rod photoreceptors in retinal slice and isolated cell preparations. Adenosine inhibited both I(Ca) and the [Ca(2+)]i increase evoked by depolarization in a dose-dependent manner with approximately 25% inhibition at 50 microM. An A2-selective agonist, (N(6)-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine) (DPMA), but not the A1- or A3-selective agonists, (R)-N(6)-(1-methyl-2-phenylethyl)adenosine and N(6)-2-(4-aminophenyl)ethyladenosine, also inhibited I(Ca) and depolarization-induced [Ca(2+)]i increases. An inhibitor of protein kinase A (PKA), Rp-cAMPS, blocked the effects of DPMA on both I(Ca) and the depolarization-evoked [Ca(2+)]i increase in rods. The results suggest that activation of A2 receptors stimulates PKA to inhibit L-type Ca(2+) channels in rods resulting in a decreased Ca(2+) influx that should suppress glutamate release.
Journal of Neurophysiology 02/2002; 87(1):351-60. · 3.32 Impact Factor
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ABSTRACT: Activation of D2-like dopamine receptors in rods with quinpirole stimulates L-type calcium currents (ICa). This result appears inconsistent with studies showing that D2-like dopamine receptor activation diminishes rod signals in second-order retinal neurons. Since small reductions in [Cl-] can inhibit photoreceptor ICa, we tested the hypothesis that enhancement of ICa with the D2/D4 receptor agonist, quinpirole, increases calcium-activated chloride currents (ICl(Ca)) causing an efflux of Cl- from rods that would provide a negative feedback inhibition of ICa. In agreement with studies from Xenopus, quinpirole reduced rod input to second-order neurons of tiger salamander retina without significantly altering rod voltage responses. Quinpirole also diminished the amplitude of depolarization-evoked increases in [Ca2+]i measured with Fura-2 in rods, a finding consistent with inhibition of synaptic transmission from rods. Electrophysiological and Cl(-)-imaging experiments indicated ECl in rods is approximately -20 mV. Quinpirole enhanced ICl(Ca) and elicited an efflux of Cl- at the resting potential. A similar Cl- efflux was produced by extracellular replacement of 24 mM Cl- with CH3SO4- and this low Cl- solution inhibited Ca2+ responses to a similar degree as quinpirole did. When ICl(Ca) was inhibited with niflumic acid, quinpirole enhanced both ICa and depolarization-evoked increases in [Ca2+]i. Furthermore, with niflumic acid, quinpirole no longer inhibited rod inputs into horizontal and bipolar cells. These results suggest an initial enhancement of ICa by quinpirole is followed by a stimulation of Cl- currents, including ICl(Ca). The net result is a Cl- efflux that inhibits depolarization-evoked increases in [Ca2+]i and synaptic transmission from rods.
Visual Neuroscience 19(3):235-47. · 2.23 Impact Factor
